A new 13-lipoxygenase from Taxus media callus is functionally linked to paclitaxel biosynthesis

The enzyme 13-Lipoxygenase (13-LOX) plays a pivotal role in the biosynthesis of jasmonates. While Taxus sp. cell suspension cultures have displayed the crucial involvement of Jasmonic acid (JA) in paclitaxel (PTX) biosynthesis activation, 13-LOX had not been identified in this plant species. In this work, a complete cDNA, encoding an 891-amino acid protein sharing high identity with other plant 13-LOXs, was identified (NCBI Genbank MW030627). The purified recombinant TmLOX13 demonstrated optimal enzymatic activity at pH 6.5 and 45 °C, displaying a strong specificity for α-linolenic acid (C18:3) and regioselectivity for carbon 13, producing 13-Hydroperoxyoctadeca-9,11,15-trienoic acid (13-HPOT). Furthermore, TmLOX13 expression in T. media cell suspension cultures peaked before the PTX production phase, suggesting a potential biological link between the 13-LOX pathway and PTX biosynthesis. This connection was validated through a small interference RNA (siRNA)-silencing approach, where silencing of TmLOX13 in T. media cells led to a significant deficiency in PTX biosynthesis, accompanied by a marked down-regulation of PTX gene expression. Similar effects were observed when the 13-LOX activity was inhibited using nordihydroguaiaretic acid (NDGA), a specific inhibitor of 13-LOX. Of particular interest, the PTX-deficient phenotype of TmLOX13-silenced cells was effectively rescued by exogenous supplementation with oxylipins derived from the 13-LOX-peroxygenase pathway. This comprehensive investigation sheds light on the functional significance of TmLOX13 in the intricate network of biochemical pathways underlying PTX biosynthesis in Taxus media.